TCR interactions with self-peptide-MHC complexes in the thymus control the key developmental check points of positive selection, lineage commitment and negative selection that are essential for the generation of a functional CD8 T cell repertoire. The mechanisms underlying these various steps are still incompletely understood. It was originally assumed that the MHC I-presented self-peptides were identical at all the selection check points. However, an exciting discovery was that cortical thymic epithelial cells (cTEC) express a unique proteasome active site subunit, ss5t, which incorporates into proteasomes to generate thymoproteasomes. Genetic inactivation of ss5t reduces the development of CD8 T cells. cTECs are the key cells that present peptide-MHC complexes for positive selection and the prevailing opinion in the field is that the role of thymoproteasomes is to generate peptides that are more optimal for this selection step. An alternate and disfavored model is that thymoproteasomes are not required to generate special peptides for positive selection but rather to make different peptides from those involved in negative selection and thereby prevent negative selection from cancelling out positive selection. So far investigators have used partial genetic knock outs to try to elucidate the role of thymoproteasomes in positive and negative selection. We have generated a more complete genetic model in which mice lack all specialized proteasomes and turn out to have a significantly stronger block in CD8 T cell development. Compared to earlier models, these mice lack all three thymoproteasome subunits in cTECs are also completely unable to switch peptides between the positive and negative selection steps. We propose to use our novel model together with other state of the art genetic models, culture systems, and mass spectrometry to elucidate the underlying mechanisms of thymic selection. Our preliminary data show feasibility and suggest that the currently favored model may be incorrect. This proposal has 3 aims to test these elucidate these issues.